Methods previously known for transformation of monocotyledons such as maize and rice, which are major grain crops, include electroporation, particle gun transformation, etc. However, these physical gene transfer methods have problems in that genes are introduced as multiple copies or are not inserted in an intact state, and the resulting transformed plants may often develop malformations and sterility.
Agrobacterium-mediated gene transfer is universally used as a transformation method for dicotyledons. Although it has been understood that hosts of Agrobacterium are limited only to dicotyledons and Agrobacterium has no ability to infect monocotyledons (Non-patent Publication No. 1), some attempts have been made to transform monocotyledons through Agrobacterium-mediated method.
Grimsley et al. have reported that when maize streak virus DNA was inserted into T-DNA of Agrobacterium and inoculated into maize growing points, infection with maize streak virus was confirmed. Since such infection symptoms are not observed simply when the maize streak virus DNA alone is inoculated, Grimsley et al. have recognized that the above observation indicates the ability of Agrobacterium to introduce DNA into maize (Non-patent Publication No. 2). However, this result is not indicative of T-DNA integration into nuclei, because a virus will multiply even when not integrated into a nuclear genome. Grimsley et al. have further demonstrated that the highest infection efficiency is observed upon inoculation into a growing point in the shoot apex of maize (Non-patent Publication No. 3), and that the VirC gene in plasmids of Agrobacterium is essential for infection (Non-patent Publication No. 4).
Gould et al. injured maize growing points with a needle and then inoculated these growing points with super-virulent Agrobacterium EHA1 carrying the kanamycin resistance gene and the GUS gene, followed by kanamycin selection on the treated growing points to obtain a resistant plant. Upon Southern analysis to confirm whether progeny seeds of this plant have the introduced gene, they confirmed that some seeds had the transgene (Non-patent Publication No. 5). This indicates that the whole plant obtained by kanamycin selection on Agrobacterium-treated growing points had both transformed and non-transformed cells (chimerism).
Mooney et al. attempted to introduce the kanamycin resistance gene into wheat embryos by using Agrobacterium. First, the embryos were enzymatically treated to injure their cell walls, and then inoculated with Agrobacterium. Among the treated calli, very few calli were grown that appeared to be resistant to kanamycin, but no whole plant was regenerated from these calli. Upon Southern analysis to confirm the presence of the kanamycin resistance gene, all the resistant calli were found to have a structural mutation in the transgene (Non-patent Publication No. 6).
Raineri et al. performed super-virulent Agrobacterium A281 (pTiBo542) treatment on 8 varieties of rice whose embryonic disc had been injured, and they confirmed tumorous tissue growth in 2 varieties of Nipponbare, Fujisaka 5. Further, when rice embryos were inoculated with Agrobacterium carrying a Ti plasmid modified to have the kanamycin resistance gene and the GUS gene wherein hormone synthesis genes in T-DNA have been removed, the growth of kanamycin-resistant calli was observed. In these resistant calli, GUS gene expression was observed, but no transformed plant was obtained. Based on these results, Raineri et al. have recognized that the Agrobacterium T-DNA was introduced into rice cells (Non-patent Publication No. 7).
As shown above, there are study reports suggesting that Agrobacterium-mediated gene transfer is also possible for Gramineae crops including rice, maize and wheat, but these reports failed to show persuasive results because these studies had a problem in reproducibility and were also insufficient for transgene confirmation (Non-patent Publication No. 8).
Chan et al. injured immature rice embryos, which had been cultured for 2 days in the presence of 2,4-D, and then inoculated these embryos with Agrobacterium carrying genes for npt II and GUS in a medium containing suspension-cultured potato cells. They cultured the thus treated immature embryos on a G418-containing medium to obtain regenerated plants from the induced calli. They confirmed the location of the GUS gene in the regenerated plants and their progeny plants by Southern analysis, and reported that the presence of the transgene was observed in plants of both R0 and R1 generations (Non-patent Publication No. 9). This result supports Agrobacterium-mediated transformation in rice, but the transformation efficiency was as low as 1.6%. Moreover, there was only one regenerated plant that showed normal growth, although 250 immature embryos were used for testing. Since enormous efforts are required to extract immature embryos of rice, such low transformation efficiency is not practical.
In recent years, it has been reported that stable and highly efficient transformation is also possible in monocotyledons including rice and maize when using a super-binary vector carrying a part of the virulence gene from super-virulent Agrobacterium (Non-patent Publications No. 10 and 11). These reports suggest that Agrobacterium-mediated transformation not only allows stable and highly efficient transformation, but is also advantageous in that the resulting transformed plants have fewer mutations, and in that the introduced genes are low in copy number and are often in an intact state. Following success in rice and maize, further reports were issued for Agrobacterium-mediated transformation in other major grain crops, i.e., wheat (Non-patent Publication No. 12), barley (Non-patent Publication No. 13) and sorghum (Non-patent Publication No. 14).
Ishida et al. (1996) (Non-patent Publication No. 11) used maize inbred lines as materials to perform Agrobacterium-mediated transformation. Thereafter, further reports were issued for Agrobacterium-mediated transformation in maize (Non-patent Publications No. 15-21). Attempts which have been made to improve the efficiency of Agrobacterium-mediated maize transformation include: selection of transformed cells on N6 basal medium (Non-patent Publication No. 21); addition of AgNO3 and carbenicillin to culture medium (Non-patent Publications No 20 and 22); and addition of cysteine to coculture medium (Non-patent Publication No. 21). Ishida et al. (2003) (Non-patent Publication No. 22) have reported that the transformation efficiency in maize is improved when cocultured immature maize embryos are selected on a medium containing AgNO3 and carbenicillin.
As shown above, in the case of Agrobacterium-mediated maize transformation, modifications to the medium composition or selection marker genes also result in improved efficiency and an extended range of varieties to be applied. However, in almost all the methods previously reported for Agrobacterium-mediated maize transformation, a transformed plant is obtained through the steps of inducing a callus from an inoculated immature embryo, allowing a transformed callus to selectively grow on a medium containing a herbicide component or an antibiotic, and placing the resulting transformed cell aggregate onto a regeneration medium (Non-patent Publications No. 15-21, 23 and 24). In some reports, tissue culture in a dedifferentiated state (callus) extends over a long period of time, more than 2 months (Non-patent Publications No. 20 and 25). Since long-term subculture in a dedifferentiated state is more likely to cause somatic cell mutations, if it is possible to obtain a regenerated plant within a minimum period of time, the resulting transformed plant can be expected to remain in a state closer to its original variety, which provides a great merit in conducting test studies and/or new variety breeding. Moreover, techniques enabling the production of transformed plants within a short period of time contribute to efficient implementation of test studies and/or new variety breeding.
Further, when performing a selection step on dedifferentiated tissues, it is necessary to excise a drug-resistant and good-shaped callus under a microscope with a surgical knife and a pair of tweezers, and then place this callus onto a fresh selective medium and a regeneration medium. Such manipulations are complicated and time-consuming, and also require a great deal of skill in selecting a good-shaped callus. Such a selection step on a selective medium extends over a long period of time and involves complicated manipulations. Moreover, culture in an undifferentiated state is more likely to cause mutations. Despite these demerits, the selection step is regarded as essential for obtaining a transformed whole plant.
In planta transformation used in Arabidopsis is a technique that can be expected to cause fewer mutations because it does not involve any culture step. However, this technique is difficult to apply in monocotyledons such as maize and rice. On the other hand, there is also a report on the production of transformed plant through Agrobacterium inoculation into a shoot apex tissue. However, such a technique has problems in efficiency and reproducibility, and hence is not practical.
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